2,2'-Dithiobis(N-ethyl-spermine-5-carboxamide) Is a High Affinity, Membrane-impermeant Antagonist of the Mammalian Polyamine Transport System

doi:10.1074/jbc.271.44.27556

Volume 271, Number 44, Issue of November 1, 1996 pp. 27556-27563
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

2,2 $'$ -Dithiobis(N-ethyl-spermine-5-carboxamide) Is a High Affinity, Membrane-impermeant Antagonist of the Mammalian Polyamine Transport System

(Received for publication, June 21, 1996, and in revised form, August 13, 1996)

Maria Huber , Joële G. Pelletier , Krikor Torossian , Patricia Dionne , Isabelle Gamache , René Charest-Gaudreault ^§ , Marie Audette and Richard Poulin

From the Laboratory of Molecular Endocrinology, Laval University Medical Research Center, 2705 Laurier Blvd., Ste. Foy, Québec, Québec, Canada G1V 4G2 and ^§ Hôpital Saint-François d'Assise Research Center, 10 Rue de l'Espinay, Québec, Québec, Canada G1L 3L5

We have synthesized 2,2 $'$ -dithiobis(N-ethyl-spermine-5-carboxamide) (DESC), its thiol monomer (MESC), and the mixed MESC-cysteamine disulfide (DEASC) as potential inhibitors of polyamine transport in mammalian cells. DESC was the most potent antagonist of spermine transport in ZR-75-1 human breast cancer cells, with K_i values of 5.0 ± 0.7, 80 ± 31, and 16 ± 3 µM for DESC, MESC, and DEASC, respectively. DESC also strongly blocked putrescine and spermidine uptake in ZR-75-1 cells (K_i = 1.6 ± 0.5 and 2.7 ± 1.1 µM, respectively). While DESC and MESC were purely competitive inhibitors of putrescine transport, DEASC was a mixed competitive/noncompetitive antagonist. Remarkably, DESC was virtually impermeant in ZR-75-1 cells despite its low K_i toward polyamine transport. The marked difference in affinity between DESC and MESC was essentially due to the tail-to-tail juxtaposition of two spermine-like structures, suggesting that dimeric ligands of the polyamine transporter might simultaneously interact with more than one binding site. While DESC strongly decreased the initial rate of [³H]spermidine transport, even a 40-fold molar excess of antagonist could not completely abolish intracellular spermidine accumulation. Moreover, as little as 0.3 µM spermidine fully restored growth in ZR-75-1 cells treated with an inhibitor of polyamine biosynthesis in the presence of 50 µM DESC, thus emphasizing the importance of uptake of trace amounts of exogenous polyamines. Thus, reducing the exogenous supply of polyamines with a potent competitive inhibitor may be kinetically inadequate to block replenishment of the polyamine pool in polyamine-depleted tumor cells that display high transport capacity. These results demonstrate that polyamine analogues cross-linked into a dimeric structure such as DESC interact with high affinity with the mammalian polyamine carrier without being used as substrates. These novel properties provide a framework for the design of specific irreversible inhibitors of the polyamine transporter, which should present advantages over competitive antagonists for an efficient blockade of polyamine transport in tumor cells.

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